Isolated Sclerotinia Sclerotiorum Of Brassica Napus Gene By Suppression Subtractive Hybridizatin

Sclerotinia Sclerotiorum is a major worldwide diseases of rape, is the first disease of three diseases of rape in China, it has called the cancer in the rape. In order to understand the rape's resistance mechanism to Sclerotinia Sclerotiorum, improve the yield of rapeseed, and breed out the best varieties of oilseed rape resistance to Sclerotinia sclerotiorum, this article is attempt to find the higher resistant varieties of rape from the respects of morphology and perspective of molecular biology, and then find some different gene fragments between NILs. At last breeding out the resistant varieties of rape germplasm.1.Using the method of toothpick vaccination to identify seven varieties rapeseed in the field, compared the morbidity, the disease index, the resistances index, and the result showed that using xiangyoul5 as the reference variety, the 98c40xxiangyoul5 and 98c40xM004 are two best varieties resistance to Sclerotinia Sclerotiorum, and the 98c40 is the susceptible variety to Sclerotinia Sclerotiorum.2. Using the method of Suppression Subtractive Hybridization, and 98c40,98c40xxiangyoul5 near-isogenic lines as the materials, constructed a cDNA gene library of rape resistance to Sclerotinia sclerotiorum. After the T-A cloning we carried out 471 single-colony PCR detection and got 342 positive bacteria. Then we chose 274 longer fragments from the library to sequence. After removing the primer sequences and repeat sequences, we submitted them to the GenBank to compare and analysis, we got 207 valid sequences in the end. These sequences include energy metabolism(10.6%), cell structure(35.7%), protein synthesis(24.6%), signal transduction(8.2%), disease resistance and defenses(20.8%), and so on.3. We picked thirteen fragments from the obtained differentially expressed fragments, according to their sequences, using Primer Premier5 software to design the primer, Then reverse transcription PCR (RT-PCR) analysis. At last we got seven fragments which involved heat shock protein, nitrilase, aldehyde oxidase, triazine-resistance, and so on.